Fluorescence resonance energy transfer screening assay for the identification of compounds that are capable of abrogating macrophage-tropic HIV-1 cell fusion

ABSTRACT

Previous studies of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein-mediated membrane fusion have focused on laboratory-adapted T-lymphotropic strains of the virus. The goal of this application was to develop a novel screening assay to characterize membrane fusion mediated by a primary HIV-1 isolate in comparison with a laboratory-adapted strain. To this end, a novel fusion assay was developed on the basis of the principle of resonance energy transfer, using HeLa cells stably transfected with gp120/gp41 from the T-lymphotropic isolate HIV-1 LA1  or the macrophage-tropic primary isolate HIV-1 JR-FL . These cells fused with CD4 +  target cell lines with a tropism mirroring that of infection by the two viruses. Of particular note, HeLa cells expressing HIV-1 JR-FL  gp120/gp41 fused only with PM1 cells, a clonal derivative of HUT 78, and not with other T-cell or macrophage cell lines. These results demonstrate that the envelope glycoproteins of these strains play a major role in mediating viral tropism. Despite significant differences exhibited by HIV-1 JR-FL  and HIV-1 LAI  in terms of tropism and sensitivity to neutralization by CD4-based proteins, the present study found that membrane fusion mediated by the envelope glycoproteins of these viruses had remarkably similar properties. In particular, the degree and kinetics of membrane fusion were similar, fusion occurred at neutral pH and was dependent on the presence of divalent cations. The claimed invention will facilitate the screening and identification of novel agents that are capable of inhibiting these interactions.

This application is a National Stage entry of International ApplicationNo. PCT/US96/09894, filed Jun. 7, 1996, which is a Continuation-In-Partof application U.S. Ser. No. 08/475,515, filed Jun. 7, 1995, the contentof which is hereby incorporated into this application by reference.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced. Thedisclosure of these publications is hereby incorporated by referenceinto this application to describe more fully the art to which thisinvention pertains.

HIV infects primarily helper T lymphocytes andmonocytes/macrophages—cells that express surface CD4—leading to agradual loss of immune function which results in the development of thehuman acquired immune deficiency syndrome (AIDS). The initial phase ofthe HIV replicative cycle involves the high affinity interaction betweenthe HIV exterior envelope glycoprotein gp120 and the cellular receptorCD4 (Klatzmann, D. R., et al., Immunodef. Rev. 2, 43-66 (1990)).Following the attachment of HIV to the cell surface, viral and targetcell membranes fuse, resulting in the introduction of the viral genomeinto the cytoplasm. Several lines of evidence demonstrate therequirement of this interaction for viral infectivity. In vitro, theintroduction of a functional cDNA encoding CD4 into human cells which donot normally express CD4 is sufficient to render these otherwiseresistant cells susceptible to HIV infection (Maddon, P. J., at al.,Cell 47,1 333-348 (1986)).

Characterization of the interaction between HIV gp120 and CD4 has beenfacilitated by the isolation of cDNA clones encoding both molecules(Maddon, P. J., et al., Cell 42, 93-104 (1985), Wain-Hobson, S., et al.,Cell 40, 9-17 (1985)). CD4 is a nonpolymorphic, lineage-restricted cellsurface glycoprotein that is a member of the immunoglobulin genesuperfamily. High-level expression of both full-length and truncated,soluble versions of CD4 (sCD4) have been described in stable expressionsystems. The availability of large quantities of purified sCD4 haspermitted a detailed understanding of the structure of this complexglycoprotein. Mature CD4 has a relative molecular weight of 55,000 andconsists of an amino-terminal 372 amino acid extracellular domaincontaining four tandem immunoglobulin-like regions denoted V1-V4,followed by a 23 amino acid transmembrane domain and a 38 amino acidcytoplasmic segment. Experiments using truncated sCD4 proteinsdemonstrate that the determinants of high-affinity binding to HIV gp120lie within the amino-terminal immunoglobulin-like domain V1 (Arthos, J.,et al., Cell 57, 469-481 (1989)). Mutational analysis of V1 has defineda discrete gp120-binding site (residues 38-52 of the mature CD4 protein)that comprises a region structurally homologous to the secondcomplementarity-determining region (CDR2) of immunoglobulins (Arthos,J., et al., Cell 57, 469-481 (1989)).

The HIV-1 envelope gene env encodes an envelope glycoprotein precursor,gp160, which is cleaved by cellular proteases before transport to theplasma membrane to yield gp120 and gp41. The membrane-spanningglycoprotein, gp41, is non-covalently associated with gp120, a purelyextracellular glycoprotein. The mature gp120 molecule is heavilyglycosylated (approximately 24 N-linked oligosaccharides), containsapproximately 480 amino acid residues with 9 intra-chain disulfide bonds(Leonard, C. K., et. al., J. Biol. Chem. 265, 10373-10382 (1990)), andprojects from the viral membrane as a dimeric or multimeric molecule(Earl, P. L., et. al. Proc. Natl. Acad. Sci. U.S.A. 87, 648-652 (1990)).

Mutational studies of HIV-1 gp120 have delineated important functionalregions of the molecule. The regions of gp120 that interact with gp41map primarily to the N- and C-termini (Helseth, E., et. al., J. Virol.65, 2119-2123 (1991)). The predominant strain-specific neutralizingepitope on gp120 is located in the 32-34 amino acid residue thirdvariable loop, herein referred to as the V3 loop, which resides near thecenter of the gp120 sequence (Bolognesi, D.P. TIBTech 8, 40-45 (1990)).The CD4-binding site maps to discontinuous regions of gp120 that includehighly conserved or invariant amino acid residues in the second, third,and fourth conserved domains (the C2, C3 and C4 domains) of gp120(Olshevsky, U., et al. J. Virol. 64, 5701-5707 (1990)). It has beenpostulated that a small pocket formed by these conserved residues withingp120 could accommodate the CDR2 loop of CD4, a region defined bymutational analyses as important in interacting with gp120 (Arthos, J.,et al., Cell 57, 469-481 (1989)).

Following the binding of HIV-1 gp120 to cell surface CD4, viral andtarget cell membranes fuse, resulting in the introduction of the viralcapsid into the target cell cytoplasm (Maddon, P. J. et al., Cell 54:865(1988)). Most evidence to date indicates that HIV-1 fusion ispH-independent and occurs at the cell surface. The HIV-1 fusion proteinis gp41, the transmembrane component of the envelope glycoprotein. Thisprotein has a hydrophobic fusion peptide at the amino-terminus andmutations in this peptide inhibit fusion (Kowalski, M. et al., Science237:1351 (1987)). In addition to gp41, recent observations suggest thatgp120 plays a role in membrane fusion distinct from its function inattachment. For example, antibodies to the principle neutralizingepitope on gp120, the V3 loop, can block infection without inhibitingattachment (Skinner, M. A. et al., J. Virol. 62:4195 (1988)). Inaddition, mutations in the tip of this loop reduce or prevent syncytiaformation in HeLa-CD4 cells expressing the mutated gp120/gp41 molecules(Freed, E. O. et al., J. Virol. 65:190 (1991)).

Several lines of evidence have implicated molecules in addition to CD4and gp120/gp41 in HIV-1 induced membrane fusion. For example, recentstudies have indicated that human cells may contain an accessorymolecule, not present in non-primate cells, which is required for HIV-1fusion (Dragic, T. et al., J. Virol. 66:4794 (1992)). The nature of thisaccessory molecule or molecules is unknown. While some studies havepostulated it might be a cell surface protease (Hattori, T. et al.,Febs. Lett. 248:48 (1989)), this has yet to be confirmed.

Fusion of the HIV-1 virion with the host cell plasma membrane ismimicked in many ways by the fusion of HIV-1 infected cells expressinggp120/gp41 with uninfected cells expressing CD4. Such cell-to-cellfusion results in the formation of multinucleated giant cells orsyncytia, a phenomenon observed with many viruses which fuse at the cellsurface. Much of our current understanding of HIV-1-induced membranefusion is derived from studies of syncytium formation. For example, thisapproach was used to demonstrate that expression of HIV-1 gp120/gp41 ina membrane, in the absence of any other viral protein, is necessary andsufficient to induce fusion with a CD4⁺ membrane (Lifson, J. D. et al.,Nature 323:725 (1986)).

Compared with virion fusion to cells, syncytium formation induced byHIV-1 appears to involve an additional step. First, thegp120/gp41-bearing membrane fuses with the CD4− bearing membrane. Thisis a rapid and reversible process which connects the membranes atlocalized sites and allows membrane-bound dyes to flow from one cell tothe other (Dimitrov, D. et al., AIDS Res. Human Retroviruses 7:799(1991)). This step presumably parallels the attachment of a virion to aCD4⁺ cell and the fusion therebetween. The second stage in cells fusionis the irreversible fusion of cells to form syncytia. The efficiency ofthis process is increased by the interaction of cellular adhesionmolecules such as ICAM-1 and LFA-1, although these molecules are notabsolutely required for syncytium formation to proceed (Golding, H. etal., AIDS Res. Human Retroviruses 8:1593 (1992)).

Most of the studies of HIV-1 fusion, including those discussed above,have been performed with strains of HIV-1 which have been extensivelypropagated in transformed human T cell lines. These strains, known aslaboratory-adapted strains, differ in several important characteristicsfrom primary or clinical isolates of the virus obtained from HIV-1infected individuals (O'Brien, W. A. et al., Nature 348:69 (1990)). Someexamples of these differences are listed in the table below.

Laboratory adapted Primary Strains Isolates tropic for transformed manyare tropic for T cell lines, do not primary monocytes and do infectprimary monocytes not infect transformed T cell lines very sensitive torelatively insensitive to neutralization by neutralization by sCD4soluble CD4 gp120 spontaneously little spontaneous stripping dissociatesfrom gp41, and sCD4 only causes and this stripping is stripping at 4°C., not increased by sCD4 at 37° C.

These differences are mirrored by differences in the primary sequence ofthe viral proteins, and in particular of the envelope glycoproteins. Insome cases, the different tropisms of primary isolates andlaboratory-adapted strains of HIV-1 have been mapped to regions on gp120such as the V3 loop (O'Brien, W. A. et al., Nature 348:69 (1990)). It ispossible that different V3 loops interact with different accessorymolecules on T cell lines or monocytes, thereby mediating tropism.

HIV-1 envelope-mediated cell fusion is a model for the early stages ofHIV-1 infection and can be used as an assay for anti-viral moleculeswhich block HIV-1 attachment and fusion (Sodroski, J. et al., Nature322-470 (1986), Lifson, J. D. et al., Nature 323:725 (1986)). Moreover,HIV-1 induced cell fusion is important in its own right as a potentialmechanism for the pathogenesis of HIV-1 infections. It is a mode oftransmission of HIV-1 from infected to uninfected cells (Gupta, P. etal., J. Virol. 63:2361 (1989), Sato, H. et al., Virology 186:712 (1992))and by this mechanism, it could contribute to the spread of HIV-1throughout the body of the infected individual. Cell fusion is also adirect mechanism of HIV-1-induced cell death (Sodroski, J. et al.,Nature 322:470 (1986), Lifson, J. D. et al., Nature 323:725 (1986)).Syncytia are seen in vivo, notably in the brains of AIDS patientssuffering from neurological complications such as AIDS dementia complex(Pumarola-Sune, T. et al., Ann. Neurol. 21:490 (1987)). In addition,syncytia have been observed in the spleens of HIV-1-infected individuals(Byrnes, R. K. et al., JAMA 250:1313 (1983)). It is possible that cellfusion may play a role in the depletion of CD4⁺ T lymphocytes that ischaracteristic of the pathogenic process leading to AIDS (Haseltine, W.A. in AIDS and the new viruses, Dalgleish, A. G. and Weiss, R. A. eds.(1990)).

In this context, it may be significant that HIV-1 isolates fromasymptomatic HIV-1-infected individuals often infect cells in vitrowithout inducing syncytia. In contrast, clinical isolates from patientswith ARC and AIDS are commonly highly virulent, syncytia-inducingstrains (Tersmette, M. et al., J. Virol. 62:2026 (1988)). In addition,there is often a switch from non-syncytium inducing (NSI) tosyncytium-inducing (SI) isolates within patients as the diseaseprogresses and symptoms appear (Tersmette, M. et al., J. Virol. 63:2118(1989), Cheng-Mayer, C. et al., science 240:80 (1988)). It is not clearwhy some HIV-1 strains do not induce syncytia, although it is possiblethat cells infected with these strains do not express sufficient levelsof gp120/gp41 for cell fusion to occur, by analogy with some otherfusogenic viruses. However, it is believed that this switch from NSI toSI HIV-1 strains influences the clinical course of HIV-1 infection. Thepresence of naturally occurring anti-syncytia antibodies in somesubjects may delay the development of HIV-1 related diseases in thesesubjects (Brenner, T. J. et al., Lancet 337:1001 (1991)).

The development of methods for measuring HIV-1 envelopeglycoprotein-mediated membrane fusion serves a useful role in furtherelucidating the mechanism of HIV-1 infection, and enabling theidentification of agents which alter HIV-1 envelopeglycoprotein-mediated cell fusion. At present there exist severalpotential methods for measuring such fusion.

The first is an assay of HIV-1 envelope glycoprotein-mediated cellfusion in which fusion is measured microscopically by measuring thetransfer of fluorescent dyes between cells (Dimitrov, D. S., et al.,AIDS Res. Human Retroviruses 7: 799-805 (1991)). This technique measuresdye distribution rather than fluorescence intensity and as such cannotbe performed using fluorometer. The assay would not be easily automatedand has not been performed with cells which stably express the HIV-1envelope glycoprotein.

The second is an assay for HIV-1 envelope-mediated cell fusion measuredbetween (a) cells which stably express the HIV-1 tat gene product inaddition to gp120/gp41, and (b) CD4⁺ cells which contain a constructconsisting of the β-galactosidase gene under the control of the HIV-1LTR promotor. When these cells fuse, β-galactosidase is expressed andcan be measured using an appropriate soluble or insoluble chromogenicsubstrate (Dragic, T., et al., Journal of Virology 66:4794 (1992)). Thisassay takes at least 1 day to perform and cannot easily be adapted tonew target cells such as primary macrophage cells. This assay also doesnot measure cell fusion in real time and is thus not amenable to use inanalyzing fusion kinetics.

Finally, the third is a fluorescence dequenching assay for the fusion ofHIV-1 virions to cells (Sinangil, F., et al., FEBS Letters 239:88-92(1988)). This assay requires the use of purified HIV-1 virions, and boththe purification of HIV-1 virions and the assay must be performed in acontainment facility. It would be difficult to readily isolatesufficient quantities of clinical virus isolates to perform the assay.Furthermore, this assay is more complicated and less reproducible than aRET assay using cells which stably express HIV-1 envelope glycoproteinsand CD4.

The methods of the subject invention employ a resonance energy transfer(RET) based assay which overcomes the problems inherent in theabove-identified methods for measuring HIV-1 envelopeglycoprotein-mediated membrane fusion. Specifically, the methods of thesubject invention employ a RET assay which is rapid, reproducible,quantitative, adaptable to various cell types, and relatively safe, andcan be automated.

SUMMARY OF THE INVENTION

The subject invention provides a method for determining whether an agentis capable of specifically inhibiting the fusion of an HIV-1 envelopeglycoprotein⁺ cell with an appropriate CD4⁺ cell which comprises: (a)contacting a sample containing a suitable amount of the agent with asuitable amount of the appropriate CD4⁺ cell and a suitable amount ofthe HIV-1 envelope glycoprotein⁺ cell under conditions which wouldpermit the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺cell in the absence of the agent, the cell membranes of the CD44 celland the HIV-1 envelope glycoprotein⁺ cell being labeled with a first dyeand a second dye, respectively, which first and second dyes permitresonance energy transfer therebetween only when juxtaposed within thesame membrane; (b) determining the percent resonance energy transfervalue of the resulting sample after a suitable period of time; (c)comparing the percent resonance energy transfer value so determined witha known standard, so as to determine whether the agent is capable ofinhibiting fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺cell; and (d) determining whether the agent inhibits the fusion of afirst control cell with a second control cell under conditions whichwould permit non-HIV-1 envelope glycoprotein-mediated fusion of thefirst and second control cells in the absence of the agent, so as todetermine whether the agent is capable of specifically inhibiting thefusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell.

The subject invention also provides a method for determining whether anagent is capable of specifically inhibiting the infection of a CD4⁺ cellwith HIV-1 which comprises determining whether the agent is capable ofspecifically inhibiting the fusion of a CD4⁺ cell with an HIV-1 envelopeglycoprotein⁺ cell by the method of the subject invention, so as tothereby determine whether the agent is capable of specificallyinhibiting the infection of a CD4⁺ cell with HIV-1.

The subject invention further provides a method for determining whetheran agent is capable of inhibiting the fusion of an HIV-1 envelopeglycoprotein⁺ cell with an appropriate CD4⁺ cell which comprises: (a)contacting a sample containing a suitable amount of the agent with asuitable amount of the CD4⁺ cell and a suitable amount of the HIV-1envelope glycoprotein⁺ cell under conditions which would permit thefusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell inthe absence of the agent, the cell membranes of the CD4⁺ cell and theHIV-1 envelope glycoprotein⁺ cell being labeled with a first dye and asecond dye, respectively, which first and second dyes permit resonanceenergy transfer therebetween only when juxtaposed within the samemembrane; (b) determining the percent resonance energy transfer value ofthe resulting sample after a suitable period of time; and (c) comparingthe percent resonance energy transfer value so determined with a knownstandard, so as to determine whether the agent is capable of inhibitingfusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell.

This invention also provides an agent determined by the above-describedmethod.

The subject invention further provides a method for quantitativelydetermining the ability of an antibody-containing sample to specificallyinhibit the fusion of an HIV-1 envelope glycoprotein⁺ cell with anappropriate CD4⁺ cell which comprises: (a) contacting a predeterminedamount of the antibody-containing sample with a suitable amount of theCD4⁺ cell and a suitable amount of the HIV-1 envelope glycoprotein⁺ cellunder conditions which would permit the fusion of the CD4⁺ cell with theHIV-1 envelope glycoprotein⁺ cell in the absence of theantibody-containing sample, the cell membranes of the CD4⁺ cell and theHIV-1 envelope glycoprotein⁺ cell being labeled with a first dye and asecond dye, respectively, which first and second dyes permit resonanceenergy transfer therebetween only when juxtaposed within the samemembrane; (b) determining the percent resonance energy transfer value ofthe resulting sample after a suitable period of time; (c) comparing thepercent resonance energy transfer value so determined with a knownstandard, so as to quantitatively determine the ability of theantibody-containing sample to inhibit the fusion of the CD4⁺ cell withthe HIV-1 envelope glycoprotein⁺ cell; and (d) determining whether theantibody-containing sample inhibits the fusion of a first control cellwith a second control cell under conditions which would permit non-HIV-1envelope glycoprotein-mediated fusion of the first and second controlcells in the absence of the agent, so as to quantitatively determine theability of the antibody-containing sample to specifically inhibit thefusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell.

The subject invention further provides a method for quantitativelydetermining the ability of an antibody-containing sample to inhibit thefusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺cell which comprises: (a) contacting a predetermined amount of theantibody-containing sample with a suitable amount of the CD4⁺ cell and asuitable amount of the HIV-1 envelope glycoprotein⁺ cell underconditions which would permit the fusion of the CD4⁺ cell with the HIV-1envelope glycoprotein⁺ cell in the -absence of the antibody-containingsample, the cell membranes of the CD4⁺ cell and the HIV-1 envelopeglycoprotein⁺ cell being labeled with a first dye and a second dye,respectively, which first and second dyes permit resonance energytransfer therebetween only when juxtaposed within the same membrane; (b)determining the percent resonance energy transfer value of the resultingsample after, a suitable period of time; and (c) comparing the percentresonance energy transfer value so determined with a known standard, soas to quantitatively determine the ability of the antibody-containingsample to inhibit the fusion of the CD4⁺ cell with the HIV-1 envelopeglycoprotein⁺ cell.

The subject invention further provides a method for determining thestage or clinical prognosis of an HIV-1 infection in an HIV-1-infectedsubject which comprises: (a) obtaining an antibody-containing samplefrom the HIV-1-infected subject; (b) quantitatively determining theability of the antibody-containing sample so obtained to inhibit thefusion of a CD4⁺ cell with an HIV-1 envelope glycoprotein⁺ cell by themethod of the subject invention; and (c) comparing the ability of theantibody-containing sample to inhibit the fusion of the CD4⁺ cell withthe HIV-1 envelope glycoprotein⁺ cell so determined with that of anantibody-containing sample obtained from an HIV-1-infected subjecthaving an HIV-1 infection at a known stage or having a known clinicalprognosis, so as to determine the stage or clinical prognosis of theHIV-1 infection in the HIV-1-infected subject.

The subject invention further provides a method for determining theefficacy of an anti-HIV-1 vaccination in a vaccinated,non-HIV-1-infected subject which comprises: (a) obtaining anantibody-containing sample from the vaccinated, non-HIV-1-infectedsubject; (b) quantitatively determining the ability of theantibody-containing sample so obtained to inhibit the fusion of an HIV-1envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell by the methodof the subject invention; and (c) comparing the ability of theantibody-containing sample to inhibit the fusion of the CD4⁺ cell withthe HIV-1 envelope glycoprotein⁺ cell so determined with that of anantibody-containing sample obtained from a vaccinated,non-HIV-1-infected subject for whom the anti-HIV-1 vaccination has aknown efficacy, so as to determine the efficacy of the anti-HIV-1vaccination in the vaccinated, non-HIV-1-infected subject.

The subject invention further provides a kit for determining whether anagent is capable of specifically inhibiting the fusion of an HIV-1envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell whichcomprises, in separate compartments: (a) a suitable amount of a CD⁺ cellwhose cell membrane is labeled with a first dye; (b) a suitable amountof an HIV-1 envelope glycoprotein⁺ cell whose cell membrane is labeledwith a second dye, the HIV-1 envelope glycoprotein⁺ cell being capableof fusing with the CD4⁺ cell of (a) under suitable conditions in theabsence of the agent, and the first and second dyes permitting resonanceenergy transfer therebetween only when juxtaposed within the samemembrane; (c) a suitable amount of a first control cell whose cellmembrane is labeled with the first dye; and (d) a suitable amount of asecond control cell whose cell membrane is labeled with the second dye,the second control cell being capable of non-HIV-1 envelopeglycoprotein-mediated fusion with the first control cell of (c) undersuitable conditions in the absence of the agent.

The subject invention further provides a kit for determining whether anagent is capable of inhibiting the fusion of a CD4⁺ cell with an HIV-1envelope glycoprotein⁺ cell which comprises, in separate compartments:(a) a suitable amount of a CD4⁺ cell whose cell membrane is labeled witha first dye; and (b) a suitable amount of an HIV-1 envelopeglycoprotein⁺ cell whose cell membrane is labeled with a second dye, theHIV-1 envelope glycoprotein⁺ cell being capable of fusing with the CD4⁺cell of (a) under suitable conditions in the absence of the agent, andthe first and second dyes permitting resonance energy transfertherebetween only when juxtaposed within the same membrane.

The subject invention further provides a method for determining whetheran HIV-1 isolate is syncytium-inducing which comprises: (a) obtaining asample of an HIV-1 isolate envelope glycoprotein⁺ cell whose cellmembrane is labeled with a first dye; (b) contacting a suitable amountof the sample with a suitable amount of a CD4⁺ cell under conditionswhich would permit the fusion of the CD4⁺ cell with a syncytium-inducingHIV-1 strain envelope glycoprotein⁺ cell, the cell membrane of the CD4⁺cell being labeled with a second dye which permits resonance energytransfer between the first dye only when the first and second dyes arejuxtaposed within the same membrane; (c) determining the percentresonance energy transfer value of the resulting sample after a suitableperiod of time; and (d) comparing the percent resonance energy transfervalue so determined with a known standard, so as to determine whetherthe HIV-1 isolate is syncytium-inducing.

Finally, the subject invention provides a method for determining thestage of an HIV-1 infection in an HIV-1-infected subject which comprisesdetermining by the method of the subject invention whether the HIV-1isolate with which the HIV-1 infected subject is infected is syncytiuminducing, so as to thereby determine the stage of the HIV-1 infection inthe HIV-1-infected subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Time course of fusion between HeLa-env⁺ cells and HeLa-CD4⁺ cellsmeasured by the RET assay.

FIG. 2 Blocking of fusion between HeLa-env⁺ cells and HeLa-CD4⁺ cells byOKT4a, measured using RET.

FIG. 3 Blocking of fusion between 160G7 cells and C8166 cells by sCD4,measured using RET.

FIG. 4 A comparative analysis of results of blocking experiments by twomethods using OKT4a to inhibit the fusion of HeLa-env⁺ and HeLa-CD4⁺cells.

FIG. 5 RET time course analysis. The time course of fusion betweenHeLa-env_(LAI) ⁺ and HeLa-CD4⁺ cells (open boxes) or HeLa-env_(JR-FL) ⁺and PM1 cells (closed boxes) was measured using the RET assay at variousintervals after mixing the cells.

FIG. 6 Inhibition of RET using the anti-attachment monoclonal antibodyOKT4A. % RET resulting from the fusion of HeLa-env_(LAI) ⁺ and HeLa-CD4⁺cells (open boxes) or HeLa-env_(JR-FL) ⁺ and PM1 cells (closed boxes)was measured in the presence and absence of various concentrations ofOKT4A. Percent inhibition of RET at each concentration of OTK4A wascalculated from this formula:

% inhibition of RET=[(A−B)/(A−C)]* 100.

Where A is the maximum % RET in the absence of antibody, B is the % RETfollowing incubation with OKT4A and C is the background % RET determinedusing HeLa cells in place of HeLa-env_(LAI) ⁺ or HeLa-env_(JR-FL) ⁺cells.

DETAILED DESCRIPTION OF THE INVENTION

The plasmid designated pMA243 was deposited pursuant to, and insatisfaction of, the requirements of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure with the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 under ATCC AccessionNo. 75626. The plasmid pMA243 was deposited with the ATCC on December16, 1993.

This invention provides a method for determining whether an agent iscapable of inhibiting the fusion of a macrophage-tropic primary isolateof HIV-1 to a CD4⁺ cell which comprises: (a) contacting (i) anappropriate CD4⁺ cell, which is labeled with a first dye, with (ii) acell expressing the HIV-1 envelope glycoprotein of the macrophage-tropicprimary isolate of HIV-1 on its surface, which is labeled with a seconddye, in the presence of an excess of the agent under conditionspermitting the fusion of the CD4⁺ cell to the cell expressing the HIV-1envelope glycoprotein on its surface in the absence of the agent, thefirst and second dyes being selected so as to allow resonance energytransfer between the dyes; (b) exposing the product of step (a) toconditions which would result in resonance energy transfer if fusion hasoccurred; and (c) determining whether there is a reduction of resonanceenergy transfer, when compared with the resonance energy transfer in theabsence of the agent, a decrease in transfer indicating that the agentis capable of inhibiting fusion of HIV-1 to CD4⁺ cells.

The subject invention provides a method for determining whether an agentis capable of specifically inhibiting the fusion of an HIV-1 envelopeglycoprotein⁺ cell with an appropriate CD4⁺ cell which comprises: (a)contacting a sample containing a suitable amount of the agent with asuitable amount of the appropriate CD4⁺ cell and a suitable amount ofthe HIV-1 envelope glycoprotein⁺ cell under conditions which wouldpermit the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺cell in the absence of the agent, the cell membranes of the CD4⁺ celland the HIV-1 envelope glycoprotein⁺ cell being labeled with a first dyeand a second dye, respectively, which first and second dyes permitresonance energy transfer therebetween only when juxtaposed within thesame membrane; (b) determining the percent resonance energy transfervalue of the resulting sample after a suitable period of time; (c)comparing the percent resonance energy transfer value so determined witha known standard, so as to determine whether the agent is capable ofinhibiting fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺cell; and (d) determining whether the agent inhibits the fusion of afirst control cell with a second control cell under conditions whichwould permit non-HIV-1 envelope glycoprotein-mediated fusion of thefirst and second control cells in the absence of the agent, so as todetermine whether the agent is capable of specifically inhibiting thefusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell.

This invention provides an agent determined to be capable ofspecifically inhibiting the fusion of an HIV-1 envelope glycoprotein⁺cell with an appropriate CD4⁺ cell using the above-described method.

This invention provides a therapeutic agent determined to be capable ofspecifically inhibiting the fusion of an HIV-1 envelope glycoprotein⁺cell with an appropriate cell using the above-described method.

As used herein, the term “agent” includes both protein and non-proteinmoieties. In one embodiment, the agent is a small molecule. In anotherembodiment, the agent is a protein. The protein may be, by way ofexample, an antibody directed against a portion of an HIV-1 envelopeglycoprotein, e.g., gp120. The agent may be derived from a library oflow molecular weight compounds or a library of extracts from plants orother organisms. In an embodiment, the agent is known. In a separateembodiment, the agent is not previously known.

As used herein, “capable of specifically inhibiting the fusion of anHIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell” means(a) capable of reducing the rate of fusion of a CD4⁺ cell membrane withHIV-1 envelope glycoprotein⁺ cell membrane by at least 5%, but notcapable of reducing the rate of non-CD4/HIV-1 envelopeglycoprotein-mediated cell membrane fusion, or (b) capable of reducingby at least 5% the total amount of fusion of a CD4⁺ cell membrane withHIV-1 envelope glycoprotein⁺ cell membrane occurring by the endpoint offusion, but not capable of reducing the total amount of non-CD4/HIV-1envelope glycoprotein-mediated cell membrane fusion occurring by theendpoint of fusion. As used herein, the rate of cell membrane fusionmeans the total quantity of cell membrane fused per unit of time. Asused herein, the “endpoint of fusion” means the point in time at whichall fusion of CD4⁺ cell membrane with HIV-1 envelope glycoprotein⁺ cellmembrane capable of occurring has occurred.

An example of the method of the subject invention is provided infra. Aknown amount of HIV-1 envelope glycoprotein⁺ cell is contacted with aknown amount of CD4⁺ cell together with an agent under conditions whichwould permit the fusion of Y amount of cell membrane per unit of time inthe absence of the agent, wherein Y is equal to the sum of the amountsof CD4⁺ cell membrane and HIV-1 envelope glycoprotein⁺ cell membrane,e.g., 0.5×Y CD4⁺ cell membrane+0.5×Y HIV-1 envelope glycoprotein⁺ cellmembrane. In the presence of the agent, 0.2×Y amount of cell membranefuses per unit of time. The agent is shown not to reduce the rate ofnon-CD4/HIV-1 envelope glycoprotein-mediated cell membrane fusion.Accordingly, the agent specifically inhibits the fusion of a CD4⁺ cellwith an HIV-1 envelope glycoprotein⁺ cell.

As used herein, the fusion of CD4⁺ cell membrane with HIV-1 envelopeglycoprotein⁺ cell membrane means the hydrophobic joining andintegration of CD4⁺ cell membrane with HIV-1 envelope glycoprotein⁺ cellmembrane to form a hybrid membrane comprising components of both cellmembranes, and does not mean the CD4/HIV-1 envelopeglycoprotein-mediated adhesion therebetween, which adhesion is aprerequisite for the fusion.

As used herein, the term “CD4” includes (a) native CD4 protein and (b) amembrane-bound CD4-based protein. As used herein, a membrane-boundCD4-based protein is any membrane-bound protein, other than native CD4,which comprises at least that portion of native CD4 which is requiredfor native CD4 to form a complex with the HIV-1 gp120 envelopeglycoprotein. In one embodiment, the CD4-based protein comprises aportion of a non-CD4 protein. If the CD4-based protein comprises aportion of a non-CD4 protein, then the portion of native CD4 which isrequired for native CD4 to form a complex with the HIV-1 gp120 envelopeglycoprotein is the portion of native CD4 having the amino acid sequencefrom +1 to about +179.

As used herein, the word “cell” includes a biological cell, e.g., a HeLacell, and a non-biological cell, e.g., a lipid vesicle (e.g., aphospholipid vesicle) or virion.

As used herein, a CD4⁺ cell is a cell having CD4 affixed to the surfaceof its cell membrane, wherein the appropriate CD4⁺ cell is capable ofspecifically binding to and fusing with an HIV-1 envelope glycoprotein⁺cell exposed thereto. In one embodiment, the suitable CD4⁺ cell is aCD4⁺ HeLa cell. In another embodiment, the suitable CD4⁺ cell is a PM1cell. In a further embodiment, the CD4⁺ cell is a primary human Tlymphocyte. In a still further embodiment, the CD4⁺ cell is a primaryhuman macrophage.

As used herein, an HIV-1 envelope glycoprotein⁺ cell is a cell havingHIV-1 envelope glycoprotein affixed to the surface of its cell membraneso as to permit the HIV-1 envelope glycoprotein⁺ cell to specificallybind to and fuse with an appropriate CD4⁺ cell exposed thereto. In oneembodiment, the HIV-1 envelope glycoprotein⁺ cell is an HIV-1 envelopeglycoprotein⁺ HeLa cell. In another embodiment, the HIV-1 envelopeglycoprotein⁺ cell is HIV-1.

Each HIV-1 isolate is tropic for a limited number of CD4⁺ cell types.Accordingly, in the subject invention, the fusion of a CD4⁺ cell with anHIV-1 envelope glycoprotein⁺ cell means the fusion of a CD4⁺ cell withan HIV-1 envelope glycoprotein⁺ cell, which HIV-1 envelope glycoproteincorresponds to an envelope glycoprotein from an HIV-1 isolate tropic forthe CD4⁺ cell. For example, the HIV-1 isolates_(JR-FL), JR-CSF and BaLare tropic for CD4⁺ primary human macrophages, the HIV-1 isolates LAIand IIIB are tropic for human CD4⁺ T lymphocyte cell lines and HeLa-CD4cells, and the RIV-1 isolates MN and SF-2 are tropic for human CD4⁺ Tlymphocyte cell lines. The HIV-1 isolates_(JR-FL), JR-CSF, BaL, LAI,IIIB, MN and SF-2 may also be tropic for CD4⁺ cell types other thanthose enumerated supra.

As used herein, an appropriate CD4⁺ cell line is a cell line that fuseswith the HIV-1 envelope glycoprotein⁺ cell line, such that the % RETmeasurement obtained is at least 5 fold greater than the backgroundlevel obtained using a combination of cells which do not fuse (e.g. HeLacells mixed with the CD4⁺ cell line). Moreover, the % RET obtained usingthe CD4⁺ cell line and the HIV-1 envelope glycoprotein⁺ cell line shouldbe inhibited to background levels using lug/ml OKT4A.

The suitable amounts of agent, CD4⁺ cell and HIV-1 envelopeglycoprotein⁺ cell may be determined according to methods well known tothose skilled in the art.

Conditions which would permit the fusion of the appropriate CD4⁺ cellwith the HIV-1 envelope glycoprotein⁺ cell in the absence of the agentare well known to those skilled in the art.

As used herein, a cell “labeled” with a dye means a cell having a dyeintegrated into its cell membrane, i.e., a cell having dye moleculescommingled with the lipid molecules of its cell membrane.

Resonance energy transfer is defined as follows: For juxtaposed dyes D1,having excitation and emission spectra Ex1 and Em1, respectively, andD2, having excitation and emission spectra Ex2 and Em2, respectively,wherein (a) Em1 has a higher average frequency than that of Em2 and (b)Em1 and Ex2 overlap, resonance energy transfer is the transfer ofelectromagnetic energy from D1 to D2 at a frequency within the Em1 andEx2 overlap, which resonance energy transfer (a) results from theelectromagnetic excitation of D1 at a frequency within the Ex1 spectrumand (b) causes the subsequent emission of electromagnetic energy from D2at a frequency within the Em2 spectrum. Accordingly, resonance energytransfer between D1 and D2 can be detected by exciting D1 withelectromagnetic energy at a frequency within Ex×1 and measuring thesubsequently emitted electromagnetic energy at a frequency within Em2,the emission of electromagnetic energy at a frequency within Em2indicating the occurrence of resonance energy transfer between D1 andD2.

The first and second dyes are “juxtaposed within the same membrane” ifthey are present within the same lipid membrane at a suitably shortdistance from each other, which suitably short distance may be readilydetermined by one skilled in the art.

In the subject invention, determining the percent resonance energytransfer value may be performed according to methods well known to thoseskilled in the art. In one embodiment, the percent resonance energytransfer value is determined by: (1) determining the resonance energytransfer value (RET) by subtracting from the total emission from D1 andD2 at a frequency within Em2 the electromagnetic energy emission due todirect D1 and D2 emission following excitation at a frequency within Ex1and emission at the frequency within Em2, which D1 and D2 emissions aremeasured by separately measuring the electromagnetic energy emission dueto cells labeled with each dye; and (2) determining the percentresonance energy transfer value (% RET value) by dividing the resonanceenergy transfer value obtained in step (1) by the total D2 emission atthe frequency within Em2.

The suitable period of time after which the percent resonance energytransfer value of the resulting sample is determined may be determinedaccording to methods well known to those skilled in the art.

The known standard is a percent resonance energy transfer value obtainedusing the CD4⁺ cell, the HIV-1 envelope glycoprotein⁺ cell, and an agenthaving a known ability to inhibit the fusion thereof.

In the subject invention, the first control cell and second control cellare capable of fusing with each other via non-HIV-1 envelopeglycoprotein-mediated fusion both in the presence and absence of anagent capable of inhibiting HIV-1 envelope glycoprotein-mediated fusion,and are not capable of fusing via HIV-1 envelope glycoprotein-mediatedfusion. Such cells are will known to those skilled in the art, andinclude, by way of example, HeLa cells which can be induced to fuse witheach other by incubation at 37° C. with polyethylene glycol 1000 or withSendai virus. These methods of inducing fusion of HeLa cells are wellknown to those skilled in the art.

In one embodiment, the agent is an antibody. As used in the subjectinvention, the term “antibody” includes, but is not limited to, bothnaturally occurring and non-naturally occurring antibodies.Specifically, the term “antibody” includes polyclonal and monoclonalantibodies, and antigen-binding fragments thereof. Furthermore, the term“antibody” includes chimeric antibodies, wholly synthetic antibodies,and antigen-binding fragments thereof.

In one embodiment, the first dye is a rhodamine moiety-containingmolecule and the second dye is a fluorescein moiety-containing molecule.Rhodamine moiety-containing molecules and fluorescein moiety-containingmolecules are well known to those skilled in the art.

In the preferred embodiment, the rhodamine moiety-containing molecule isoctadecyl rhodamine B chloride and the fluorescein moiety-containingmolecule is fluorescein octadecyl ester.

In another embodiment, the first dye is a fluorescein moiety-containingmolecule and the second dye is a rhodamine moiety-containing molecule.

In one embodiment, the CD4⁺ cell is a CD4⁺ HeLa cell. In anotherembodiment of the subject invention, the HIV-1 envelope glycoprotein⁺cell is an HIV-1_(LAI) gp120/gp41⁺ HeLa cell. In a separate embodiment,the CD4⁺ cell is a PM1 cell and the HIV-1 envelope glycoprotein⁺ cell isan HIV-1_(JR-FL) gp120/gp41 HeLa cell. In a further embodiment, the CD4⁺cell is a primary human T lymphocyte. In a still further embodiment, theCD4⁺ cell is a primary human macrophage.

The subject invention also provides a method for determining whether anagent is capable of specifically inhibiting the infection of a CD4⁺ cellwith HIV-1 which comprises determining whether the agent is capable ofspecifically inhibiting the fusion of a CD4⁺ cell with an HIV-1 envelopeglycoprotein⁺ cell by the method of the subject invention, so as tothereby determine whether the agent is capable of specificallyinhibiting the infection of a CD4⁺ cell with HIV-1.

The subject invention further provides a method for determining whetheran agent is capable of inhibiting the fusion of an HIV-1 envelopeglycoprotein⁺ cell with an appropriate CD4⁺ cell which comprises: (a)contacting a sample containing a suitable amount of the agent with asuitable amount of the CD4⁺ cell and a suitable amount of the HIV-1envelope glycoprotein⁺ cell under conditions which would permit thefusion of the appropriate CD4⁺ cell with the HIV-1 envelopeglycoprotein⁺ cell in the absence of the agent, the cell membranes ofthe CD4⁺ cell and the HIV-1 envelope glycoprotein⁺ cell being labeledwith a first dye and a second dye, respectively, which first and seconddyes permit resonance energy transfer therebetween only when juxtaposedwithin the same membrane; (b) determining the percent resonance energytransfer value of the resulting sample after a suitable period of time;and (c) comparing the percent resonance energy transfer value sodetermined with a known standard, so as to determine whether the agentis capable of inhibiting fusion of the HIV-1 envelope glycoprotein⁺ cellwith the CD4.

As used herein, “capable of inhibiting the fusion of an HIV-1 envelopeglycoprotein⁺ cell with an appropriate CD4⁺ cell” means capable of (a)reducing the rate of fusion of CD4⁺ cell membrane with HIV-1 envelopeglycoprotein⁺ cell membrane by at least 5%, or (b) reducing by at least5% the total amount of fusion of CD4⁺ cell membrane with HIV-1 envelopeglycoprotein⁺ cell membrane occurring by the endpoint of fusion. Anagent capable of inhibiting the fusion of an HIV-1 envelopeglycoprotein⁺ cell with an appropriate CD4⁺ cell may also be capable ofreducing the rate to non-CD4/HIV-1 envelope glycoprotein-mediated cellmembrane fusion.

This invention provides an agent determined to be capable of inhibitingthe fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriateCD4⁺ cell using the above-described method.

In one embodiment, the first dye is a rhodamine moiety-containingmolecule and the second dye is a fluorescein moiety-containing molecule.

In one embodiment, the CD4⁺ cell is a CD4⁺ HeLa cell. In anotherembodiment of the subject invention, the HIV-1 envelope glycoprotein⁺cell is an HIV-1_(LAI) gp120/gp41⁺ HeLa cell. In a separate embodiment,the CD4⁺ cell is a PM1 cell and the HIV-1 envelope glycoprotein⁺ cell isan HIV-1_(JR-FL) gp120/gp41 HeLa cell. In a further embodiment, the CD4⁺cell is a primary human T lymphocyte. In a still further embodiment, theCD4⁺ cell is a primary human macrophage.

In the preferred embodiment, the rhodamine moiety-containing molecule isoctadecyl rhodamine B chloride and the fluorescein moiety-containingmolecule is fluorescein octadecyl ester.

In another embodiment, the first dye is a fluorescein moiety-containingmolecule and the second dye is a rhodamine moiety-containing molecule.

The subject invention further provides a method for quantitativelydetermining the ability of an antibody-containing sample to specificallyinhibit the fusion of an HIV-1 envelope glycoprotein⁺ cell with anappropriate CD4⁺ cell which comprises: (a) contacting a predeterminedamount of the antibody-containing sample with a suitable amount of theappropriate CD4⁺ cell and a suitable amount of the HIV-1 envelopeglycoprotein⁺ cell under conditions which would permit the fusion of theCD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell in the absence ofthe antibody-containing sample, the cell membranes of the CD4⁺ cell andthe HIV-1 envelope glycoprotein⁺ cell being labeled with a first dye anda second dye, respectively, which first and second dyes permit resonanceenergy transfer therebetween only when juxtaposed within the samemembrane; (b) determining the percent resonance energy transfer value ofthe resulting sample after a suitable period of time; (c) comparing thepercent resonance energy transfer value so determined with a knownstandard, so as to quantitatively determine the ability of theantibody-containing sample to inhibit the fusion of the CD4⁺ cell withthe HIV-1 envelope glycoprotein⁺ cell; and (d) determining whether theantibody-containing sample inhibits the fusion of a first control cellwith a second control cell under conditions which would permit non-HIV-1envelope glycoprotein-mediated fusion of the first and second controlcells in the absence of the agent, so as to quantitatively determine theability of the antibody-containing sample to specifically inhibit thefusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell.

The antibody-containing sample may be any antibody-containing sample. Inone embodiment, the antibody-containing sample is a serum sample. Inanother embodiment, the antibody-containing sample is an IgGpreparation. Methods of obtaining an antibody-containing sample are wellknown to those skilled in the art.

In one embodiment, the first dye is a rhodamine moiety-containingmolecule and the second dye is a fluorescein moiety-containing molecule.

In the preferred embodiment, the rhodamine moiety-containing molecule isoctadecyl rhodamine B chloride and the fluorescein moiety-containingmolecule is fluorescein octadecyl ester.

In another embodiment, the first dye is a fluorescein moiety-containingmolecule and the second dye is a rhodamine moiety-containing molecule.

In one embodiment, the CD4⁺ cell is a CD4⁺ HeLa cell. In anotherembodiment of the subject invention, the HIV-1 envelope glycoprotein⁺cell is an HIV-1_(LAI) gp120/gp41⁺ HeLa cell. In a separate embodiment,the CD4⁺ cell is a PM1 cell and the HIV-1 envelope glycoprotein⁺ cell isan HIV-1_(JR-FL) gp120/gp41 HeLa cell. In a further embodiment, the CD4⁺cell is a primary human T lymphocyte. In a still further embodiment, theCD4⁺ cell is a primary human macrophage.

The subject invention further provides a method for quantitativelydetermining the ability of an antibody-containing sample to inhibit thefusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺cell which comprises: (a) contacting a predetermined amount of theantibody-containing sample with a suitable amount of the appropriateCD4⁺ cell and a suitable amount of the HIV-1 envelope glycoprotein⁺ cellunder conditions which would permit the fusion of the CD4⁺ cell with theHIV-1 envelope glycoprotein⁺ cell in the absence of theantibody-containing sample, the cell membranes of the CD4⁺ cell and theHIV-1 envelope glycoprotein⁺ cell being labeled with a first dye and asecond dye, respectively, which first and second dyes permit resonanceenergy transfer therebetween only when juxtaposed within the samemembrane; (b) determining the percent resonance energy transfer value ofthe resulting sample after a suitable period of time; and (c) comparingthe percent resonance energy transfer value so determined with a knownstandard, so as to quantitatively determine the ability of theantibody-containing sample to inhibit the fusion of the HIV-1 envelopeglycoprotein⁺ with the CD4⁺ cell.

In one embodiment, the first dye is a rhodamine moiety-containingmolecule and the second dye is a fluorescein moiety-containing molecule.

In the preferred embodiment, the rhodamine moiety-containing molecule isoctadecyl rhodamine B chloride and the fluorescein moiety-containingmolecule is fluorescein octadecyl ester.

In another embodiment, the first dye is a fluorescein moiety-containingmolecule and the second dye is a rhodamine moiety-containing molecule.

In one embodiment, the CD4⁺ cell is a CD4⁺ HeLa cell. In anotherembodiment of the subject invention, the HIV-1 envelope glycoprotein⁺cell is an HIV-1_(LAI) gp120/gp41⁺ HeLa cell. In a separate embodiment,the CD4⁺ cell is a PM1 cell and the HIV-1 envelope glycoprotein⁺-cell isan HIV-1_(JR-FL) gp120/gp41 HeLa cell. In a further embodiment, the CD4⁺cell is a primary human T lymphocyte. In a still further embodiment, theCD4⁺ cell is a primary human macrophage.

The subject invention further provides a method for determining thestage of clinical prognosis of an HIV-1 infection in an HIV-1-infectedsubject which comprises: (a) obtaining an antibody-containing samplefrom the HIV-1-infected subject; (b) quantitatively determining theability of the antibody-containing sample so obtained to inhibit thefusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺cell by the method of the subject invention; and (c) comparing theability of the antibody-containing sample to inhibit the fusion of theCD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell so determined withthat of an antibody-containing sample obtained from an HIV-1 infectedsubject having an HIV-1 infection at a known stage or having a knownclinical prognosis, so as to determine the stage or clinical prognosisof the HIV-1 infection in the HIV-1-infected subject.

As used herein, an “HIV-infected subject” means a subject having atleast one of his own cells invaded by HIV-1. In the preferredembodiment, the subject is a human.

The subject invention further provides a method for determining theefficacy of an anti-HIV-1 vaccination in a vaccinated,non-HIV-1-infected subject which comprises: (a) obtaining anantibody-containing sample from the vaccinated, non-HIV-1-infectedsubject; (b) quantitatively determining the ability of theantibody-containing sample so obtained to inhibit the fusion of an HIV-1envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell by the methodof the subject invention; and (c) comparing the ability of theantibody-containing sample to inhibit the fusion of the CD4⁺ cell withthe HIV-1 envelope glycoprotein⁺ cell so determined with that of anantibody-containing sample obtained from a vaccinated,non-HIV-1-infected subject for whom the anti-HIV-1 vaccination has aknown efficacy, so as to determine the efficacy of the anti-HIV-1vaccination in the vaccinated, non-HIV-1-infected subject.

As used herein, “anti-HIV-1 vaccination” means the administration to asubject of a vaccine intended to elicit the production of antibodies bythe vaccinated subject which are capable of specifically binding toepitopes present on an HIV-1 surface envelope glycoprotein. Vaccines ingeneral are well known to those skilled in the art, and comprise anantigen, e.g., a protein, and an adjuvant.

As used herein, the “efficacy of an anti-HIV-1 vaccination” means thedegree to which the vaccination or successive vaccinations (i.e.,immunization) causes the titre of HIV-1-neutralizing antibodies in thevaccinated subject to increase. In other words, the higher the efficacyof an anti-HIV-1 vaccination, the higher the titre of HIV-1-neutralizingantibodies in the vaccinated subject.

As used herein, a “non-HIV-1-infected subject” means a subject nothaving any of his own cells invaded by HIV-1. In the preferredembodiment, the subject is a human.

The subject invention further provides a kit for determining whether anagent is capable of specifically inhibiting the fusion of an HIV-1envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell whichcomprises, in separate compartments: (a) a suitable amount of anappropriate CD4⁺ cell whose cell membrane is labeled with a first dye;(b) a suitable amount of an HIV-1 envelope glycoprotein⁺ cell whose cellmembrane is labeled with a second dye, the HIV-1 envelope glycoprotein⁺cell being capable of fusing with the CD4⁺ cell of (a) under suitableconditions in the absence of the agent, and the first and second dyespermitting resonance energy transfer therebetween only when juxtaposedwithin the same membrane; (c) a suitable amount of a first control cellwhose cell membrane is labeled with the first dye; and (d) a suitableamount of a second control cell whose cell membrane is labeled with thesecond dye, the second control cell being capable of non-HIV-1 envelopeglycoprotein-mediated fusion with the first control cell of (c) undersuitable conditions in the absence of the agent.

The kit of the subject invention may further comprise additionalbuffers. Furthermore, the cells may either be dried or suspended inliquid or gel.

The suitable amounts of cells are amounts which would permit one skilledin the art to determine, without undue experimentation, whether an agentis capable of specifically inhibiting the fusion of a CD4⁺ cell with anHIV-1 envelope glycoprotein⁺ cell. Such amounts may be readilydetermined according to methods well known to those skilled in the art.

In one embodiment, the first dye is a rhodamine moiety-containingmolecule and the second dye is a fluorescein moiety-containing molecule.

In the preferred embodiment, the rhodamine moiety-containing molecule isoctadecyl rhodamine B chloride and the fluorescein moiety-containingmolecule is fluorescein octadecyl ester.

In another embodiment, the first dye is a fluorescein moiety-containingmolecule and the second dye is a rhodamine moiety-containing molecule.

In one embodiment, the CD4⁺ cell is a CD4⁺ HeLa cell. In anotherembodiment of the subject invention, the HIV-1 envelope glycoprotein⁺cell is an HIV-1_(LAI) gp120/gp41⁺ HeLa cell. In a separate embodiment,the CD4⁺ cell is a PM1 cell and the HIV-1 envelope glycoprotein⁺-cell isan HIV-1_(JR-FL) gp120/gp41 HeLa cell. In a further embodiment, the CD4⁺cell is a primary human T lymphocyte. In a still further embodiment, theCD4⁺ cell is a primary human macrophage.

The subject invention further provides a kit for determining whether anagent is capable of inhibiting the fusion of a CD4⁺ cell with an HIV-1envelope glycoprotein⁺ cell which comprises, in separate compartments:(a) a suitable amount of a CD4⁺ cell whose cell membrane is labeled witha first dye; and (b) a suitable amount of an HIV-1 envelopeglycoprotein⁺ cell whose cell membrane is labeled with a second dye, theHIV-1 envelope glycoprotein⁺ cell being capable of fusing with the CD4⁺cell of (a) under suitable conditions in the absence of the agent, andthe first and second dyes permitting resonance energy transfertherebetween only when juxtaposed within the same membrane.

The kit of the subject invention may further comprise additionalbuffers. Furthermore, the cells may either be dried or suspended in aliquid or gel carrier.

The suitable amounts of cells are amounts which would permit one skilledin the art to determine, without undue experimentation, whether an agentis capable of inhibiting the fusion of a CD4⁺ cell with an HIV-1envelope glycoprotein⁺ cell. Such amounts may be readily determinedaccording to methods well known to those skilled in the art.

In one embodiment, the first dye is a rhodamine moiety-containingmolecule and the second dye is a fluorescein moiety-containing molecule.

In the preferred embodiment, the rhodamine moiety-containing molecule isoctadecyl rhodamine B chloride and the fluorescein moiety-containingmolecule is fluorescein octadecyl ester.

In another embodiment, the first dye is a fluorescein moiety-containingmolecule and the second dye is a rhodamine moiety-containing molecule.

In one embodiment, the CD4⁺ cell is a CD4⁺ HeLa cell. In anotherembodiment of the subject invention, the HIV-1 envelope glycoprotein⁺cell is an HIV-1_(LAI) gp120/gp41⁺ HeLa cell. In a separate embodiment,the CD4⁺ cell is a PM1 cell and the HIV-1 envelope glycoprotein⁺-cell isan HIV-1_(JR-FL) gp120/gp41 HeLa cell. In a further embodiment, the CD4⁺cell is a primary human T lymphocyte. In a still further embodiment, theCD4⁺ cell is a primary human macrophage.

The subject invention further provides a method for determining whetheran HIV-1 isolate is syncytium-inducing which comprises: (a) obtaining asample of an HIV-1 isolate envelope glycoprotein⁺ cell whose cellmembrane is labeled with a first dye; (b) contacting a suitable amountof the sample with a suitable amount of a CD4⁺ cell under conditionswhich would permit the fusion of the CD4⁺ cell with a syncytium-inducingHIV-1 strain envelope glycoprotein⁺ cell, the cell membrane of the CD4⁺cell being labeled with a second dye which permits resonance energytransfer between the first dye only when the first and second dyes arejuxtaposed within the same membrane; (c) determining the percentresonance energy transfer value of the resulting sample after a suitableperiod of time; and (d) comparing the percent resonance energy transfervalue so determined with a known standard, so as to determine whetherthe HIV-1 isolate is syncytium-inducing.

As used herein, “syncytium-inducing” means capable of causing theformation of syncytia (multi-nucleated cells resulting from HIV-1envelope glycoprotein-mediated cell fusion) when contacted with aplurality of CD4⁺ cells under suitable conditions.

Obtaining a sample of an HIV-1 isolate envelope glycoprotein cells maybe performed according to methods well known to those skilled in theart.

HIV-1 isolate envelope glycoprotein⁺ cells may be obtained from blood orany other bodily fluid known to contain HIV-1 isolate envelopeglycoprotein⁺ cells in HIV-infected subjects. Alternatively, HIV-1isolate envelope glycoprotein⁺ cells may be obtained by culturing cellsin vitro with blood or other bodily fluids containing the HIV-1 isolateor HIV-1 isolate-infected cells, and recovering the HIV-1 isolateenvelope glycoprotein⁺ cells produced thereby.

The suitable amounts of sample and CD4⁺ cell may be determined accordingto methods well known to those skilled in the art.

In one embodiment, the first dye is a rhodamine moiety-containingmolecule and the second dye is a fluorescein moiety-containing molecule.

In the preferred embodiment, the rhodamine moiety-containing molecule isoctadecyl rhodamine B chloride and the fluorescein moiety-containingmolecule is fluorescein octadecyl ester.

In another embodiment, the first dye is a fluorescein moiety-containingmolecule and the second dye is a rhodamine moiety-containing molecule.

In one embodiment, the CD4⁺ cell is a CD4⁺ HeLa cell. In anotherembodiment, the CD4⁺ cell is a PM1 cell. In a further embodiment, theCD4⁺ cell is a primary human T lymphocyte. In a still furtherembodiment, the CD4⁺ cell is a primary human macrophage.

The subject invention further provides a method for determining thestage of an HIV-1 infection in an HIV-1-infected subject which comprisesdetermining by the method of the subject invention whether the HIV-1isolate with which the HIV-1-infected subject is infected issyncytium-inducing, so as to thereby determine the stage of the HIV-1infection in the HIV-1-infected subject.

Finally, the subject invention provides a method for quantitativelymeasuring the fusion of an HIV-1 envelope glycoprotein⁺ cell with anappropriate CD4⁺ which comprises: (a) contacting a sample of theappropriate CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell underconditions permitting fusion therebetween, the cell membranes of theCD4⁺ cell and the HIV-1 envelope glycoprotein⁺ cell being labeled with afirst dye and a second dye, respectively, which first and second dyespermit resonance energy transfer therebetween only when juxtaposedwithin the same membrane; (b) determining the percent resonance energytransfer value of the resulting sample after a suitable period of time;and (c) comparing the percent resonance energy transfer value sodetermined with a known standard, so as to quantitatively measure thefusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS

First Series of Experiments

A Background

The RET-based fusion assay of the subject invention measures fusionbetween cells which express the HIV-1 envelope glycoprotein (gp120/gp41)and cells which express CD4. Such cell-cell fusion may lead to theproduction of multinucleated cells or syncytia. Molecules which blockHIV-1 attachment or fusion to host cells also block syncytia formation.Syncytia assays have been used in many laboratories to detect virus oranti-virus molecules, and typically have a visual readout. In thedevelopment of the assay, permanent cell lines which stably expressgp120/gp41 or CD4 were used.

The resonance energy transfer technique has been used in a variety ofstudies of membrane fusion including the fusion of nucleated cellsinduced by viruses or polyethylene glycol. However, it has notpreviously been used to study HIV-1 envelope glycoprotein-mediatedmembrane fusion. The technique involves labeling one fusion partner(e.g. a gp120/gp41-expressing cell line) with a fluorescent dye such asoctadecyl fluorescein (F18) and the other fusion partner (e.g. aCD4-expressing cell line) with a dye such as octadecyl rhodamine (R18).The dyes are chosen such that the emission spectrum of one (F18)overlaps the excitation spectrum of the second (R18). When the cellsfuse, the F18 and R18 associate together closely enough that stimulationof F18 results in resonance energy transfer to R18 and emission at theR18 emission wavelengths. The octadecyl versions of the fluorsspontaneously insert into the plasma membranes of cells using thelabeling protocol described below.

B Cells Tested

(1) A Chinese Hamster Ovary (CHO) cell line which expresses HIV-1_(IIIB)gp120/gp41 (160G7) was mixed with a human T lymphocyte cell line whichexpresses CD4 (C8166). CD4⁺ cells are commercially available. 160G7cells may be obtained at the MRC AIDS Directed Program (United Kingdom).C8166 cells may be obtained at the MRC AIDS Directed Program (UnitedKingdom) and the NIH AIDS Research and Reference Reagent Program(Bethesda, Md.). It was previously demonstrated that 160G7 cells andC8166 cells fuse to form multinucleated syncytia. This assay is asyncytium assay which requires visual counting of syncytia with the aidof a low power microscope. This assay is suitable for analyzing blockingagents such as CD4-based molecules and neutralizing antibodies directedagainst gp120 and gp41.

(2) Human epithelial carcinoma (HeLa) cells which express HIV-1_(LAI)gp120/gp41 (HeLa-env) and HeLa cells which express CD4 (HeLa-CD4⁺ ) werealso used. HeLa-CD4⁺ cells may be obtained at the MRC AIDS DirectedProgram (United Kingdom) and the NIH AIDS Research and Reference ReagentProgram (Bethesda, Md.). HeLa-env⁺ cells express much higher levels ofgp120/gp41 than do 160G7 cells, as demonstrated by the ability to easilydetect gp120 on the surface of HeLa-env⁺ cells but not 160G7 cells byflow cytometry using an anti-gp120 antibody. Visual analysisdemonstrates that HeLa-env⁺ cells fuse readily with C8166 and HeLa-CD4⁺cells to form syncytia.

HeLa-env⁺ cells may be obtained, for example, by transfecting HeLa cellswith an env-encoding plasmid, such as pMA243, using the calciumphosphate precipitation method and subsequent selection of transfectantswith 2 μM methotrexate. The plasmid pMA243 is designed to express theHIV-1_(LAI) genes env, tat, rev and vpu, in addition to the selectablemarker DHFR*, with all genes under the control of the HIV-1 LTR (Dragic,T., et al., J. Virol. 66:4794-4802 (1992)). DHFR* is a mutantdihydrofolate reductase gene that demonstrates a reduced affinity formethotrexate. In pMA243, the DHFR* gene is expressed from the mRNAspliced transcript that normally encodes the HIV-1 nef gene which isdeleted in this vector. The HIV-1-encoded tat and rev genes are requiredfor high level expression of the env gene. The plasmid pMA243 alsoencodes an ampicillin resistance marker and bacterial origin ofreplication.

C Cuvette Assay Method

The cell labeling conditions were modified from those used in a previousstudy where RET was used to monitor polyethylene glycol-induced cellfusion (Wanda, P. E., and Smith, J. D., J. Histochem. Cytochem. 30:1297(1982)). F18 (fluorescein octadecyl ester; Molecular Probes Eugene,Oreg. Catalog No. F3857) or R18 (octadecyl rhodamine B, chloride salt;Molecular Probes, Catalog No. 0246) were dissolved in ethanol at 5-10mg/ml and diluted approximately 1000-fold into the appropriate growthmedium. The exact concentration in the medium was adjusted to bring theOD to 0.34 at 506 nm (F18) or 1.04 at 565 nm (R18). Monolayers of cellswere incubated with the appropriate medium overnight, then washed andcounted. 100,000 cells of each type were mixed together in wells of a24-well tissue culture plate. At intervals after mixing, the cells wereremoved with EDTA, washed and placed in a fluorometer cuvette.Fluorescence was measured at three sets of excitation and emissionwavelengths (see table 1 below) using a Perkin-Elmer LS50 fluorometer.

TABLE 1 Excitation Emission measurement wavelength wavelength obtained450 nm 530 nm Total F18 fluorescence 557 nm 590 nm Total R18fluorescence 450 nm 590 nm RET* *The calculation of RET requires firstsubtracting the fluorescence due to direct F18 and R18 fluorescencefollowing excitation at 450 and emission at 590. The fluorescencemeasurements are determined by measuring the fluorescence of cellslabeled with each dye separately.

The RET value, calculated as described above, is divided by the totalR18 fluorescence to give a % RET value. The results of initialexperiments indicate that RET can be measured using both cellcombinations listed above. A greater signal was produced when theenvelope glycoprotein-expressing cells were F18-labeled and theCD4-expressing cells were R18 labeled than when the envelopeglycoprotein-expressing cells were R18-labeled and the CD4-expressingcells were F18 labeled.

D Results of Time Course RET Studies and Experiments With Control CellLines, Using the Cuvette Assay Method

Time course experiments were performed with the HeLa-env⁺+HeLa-CD4⁺combination (FIG. 1). A control cell line, HeLa-Δenv⁺, was used.HeLa-Δenv⁺ cells express HIV-1 envelope glycoprotein, with a 400 basepair deletion in the gp120-encoding region of the env gene. These cellsdo not fuse with CD4⁺ human cells.

The results demonstrate that fusion can be measured by the RET assay at2 hours, but not at 1 hour, consistent with previous studies of HIV-1envelope-mediated cell fusion using fluorescence microscopy. At 4 hours,massive cell fusion was evident by visual inspection of the culture, andthis time point yielded reproducible RET values in several experiments.In other experiments, the combination of 160G7 cells with C8166 cellsgave a reproducible maximum RET value at about 4 hours but with lowervalues than those obtained using HeLa-env⁺ and HeLa-CD4⁺ (data notshown). Presumably, this difference results from the much greater levelof gp120/gp41 expression on HeLa-env⁺ cells as compared with 160G7cells.

A number of control experiments were performed using combinations ofcells which, based on previous studies, are known not to fuse. Thesecombinations included HeLa cells combined with HeLa-CD4⁺ cells, orHeLa-env⁺ cells combined with CHO-CD4 or the human glioma cell lineU87.MG-CD4. CHO-CD4 cells, like other non-primate cells, do not fusewith cells expressing HIV-1 gp120/gp41. U87.MG-CD4 cells are one of thefew CD4⁺ human cell lines which do not fuse with HIV-1 envelopeglycoprotein-expressing cells. RET values obtained with thesecombinations of cells (data not shown) were in general similar to thoseusing the control HeLa-Δenv⁺+HeLa-CD4⁺ (FIG. 1).

E Results of RET Experiments With Blocking Agents Using the CuvetteAssay Method

It was next determined whether sCD4 (which interacts with gp120/gp41⁺cells) or the murine MAb OXT4a (which interacts with CD4⁺ cells) couldblock RET (FIGS. 2 and 3). Both these molecules are known to inhibitHIV-1 infection and syncytium formation. The percent blocking wascalculated as t RET at each concentration of blocking agent divided by eRET in the absence of blocking agent at 4 hours.

As shown in FIGS. 2 and 3, both sCD4 and OKT4a block fusion as measuredby RET. The concentrations of these agents required for 50% inhibitionare similar to those determined using other assays. For example. theIC₅₀ for sCD4 inhibition of fusion between 160G7 an C8166 wasapproximately 4 μg/ml measured using the RET assay, as compared with 5.5μg/ml measured by a visual syncytium assay (i.e., an assay for measuringthe inhibition of syncytium formation, wherein the syncytia arequantitated visually using a low-power microscope) using the samecombination of cells. In summary, these results demonstrate that the RETmethod can be used to measure HIV-1 envelope-mediated cell fusion in arapid and reproducible fashion. When compared with data from the moreconventional visual syncytium assay, the results are in excellentagreement.

F Control Blocking Experiment With IKT4 Using Cuvette Assay Method

Control experiments were performed to examine inhibition of % RET byOKT4. OKT4 is a mouse monoclonal antibody that binds CD4 but does notinhibit the CD4-gp120 interaction, HIV-1 infection, or HIV-induced cellfusion. Using the cuvette method and the HeLa-env⁺+HeLa-CD4⁺combination, OKT4 gave 0% inhibition of RET at 0.2 μg/ml or 2.0 μg/ml,compared with 65% inhibition by OKT4a at 0.2 μg/ml in the sameexperiment. These results demonstrate that inhibition of HIV-1envelope-mediated membrane fusion as measured by RET is specific foragents that block HIV-1 infection and HIV-induced cell fusion.

G Automation of the RET Assay Using the Plate Reader Assay

A fluorescent plate reader was used to analyze the RET assay. Thismethod has the advantage of reducing the manipulations required, notablythe need to remove cells for measurement of fluorescence in a cuvette.The plate reader measures fluorescence of cells directly in a multi-welltissue culture plate. Moreover, the speed of assay readout isdramatically increased (by approximately 100-fold). The Millipore“Cytofluor” was used in this experiment. This is a dedicated platereader which has been used in a variety of different cell-basedfluorescence assays and is suitable for use with a range of plateformats including 24-well and 96-well tissue culture plates. TheCytofluor also has the major advantages of speed and compatibility withIBM software analysis programs.

The results indicate that the assay can be readily performed in 24 or 96well tissue culture plates using the fluorescence plate reader.

In one embodiment, when performing the assay on a routine basis, twotypes of measurements are done. In the first, RET is measured at asingle time point following mixing of labeled cells and a candidateblocking agent. In the second, the assay is adapted to measure changesin the rate of cell fusion in the presence or absence of blockingagents. One of the advantages of the RET assay is that it measuresfusion in real time and thus is amenable to kinetic analysis.

For example, a method of using the plate reader assay and measuring RETat a single time point is provided below. In this assay a 96-well flatbottom tissue culture plate is used. The method is a modification of thecuvette method described above.

Example of a single time-point plate reader assay method:

1. Prepare dyes:

R18: 10 mg/ml in 100! EtOH (for HeLa-CD4⁺ cells)

F18: 5 mg/ml in 100% EtOH (for HeLa-env⁺ cells)

2. Add dyes to appropriate concentrations, in cell culture mediumcontaining 10% fetal calf serum, as determined by absorbancemeasurements:

F18⁺ medium: 0.34 at 506 nm

R18⁺ medium: 0.52 at 565 nm

3. Add medium+dye to the appropriate cells as indicated above, thenincubate overnight to stain cells.

4. Wash cells and count.

5. Plate out 20,000 cells of each line/well, some wells having one orother cell line separately, other wells with both cell lines, and otherwells with various concentrations of antibodies or other inhibitoryagents added in addition to both cell lines.

6. 4 hours later, remove the media and wash all of the wells three timeswith PBS (the cells remain adherent in the wells). Add 200 μl PBS toeach well. Read fluorescence in the wells using the Millipore Cytofluorplate reader with filter combinations listed below:

F18: excitation 450 nm emission 530 nm (X)

R18: excitation 530 nm emission 590 nm (Y)

F18+R18: excitation 450 nm emission 590 nm (Z)

The emission values, X, Y and Z (as indicated above) are recorded foreach cell combination:

A) HeLa-env⁺+HeLa-CD4⁺

B) HeLa-env⁺ alone

C) HeLa-CD4⁺ alone

For example, the F18 reading for HeLa-env⁺ cells alone is given byB_(x).

Then % RET is calculated using this formula:${\% \quad {RET}} = {\frac{A_{Z} - \left( {A_{X} \cdot {B_{Z}/B_{X}}} \right) - \left( {A_{Y} \cdot \quad {C_{Z}/C_{Y}}} \right)}{A_{Y}} \cdot 100}$

Similar results were obtained in experiments comparing inhibition of %RET using the cuvette method and the plate reader method. For example,FIG. 4 illustrates the inhibition of fusion between HeLa-env⁺ andHeLa-CD4⁺ cells by the monoclonal anti-CD4 antibody, OKT4a, measured asa reduction in % RET determined by both methods at 4 hours after mixingthe cells.

Second Series of Experiments

As discussed in the first series of experiments, the properties oflaboratory-adapted strains of HIV-1 differ from those of primaryisolates of the virus. While the former infect continuous Tlymphoblastoid cell lines or other human cell lines engineered toexpress human CD4, the latter are often macrophage-tropic and usuallyinfect only primary macrophage cultures. Macrophage-tropic isolates ofHIV-1 are particularly important because they are usually the strainswhich mediate transmission from individual to individual, whether thistransmission is by sexual, parenteral or vertical routes (Zhu, T., etal., Science 261:1179(1993); van't Wout, A. B., et al. J. Clin. Invest.,94:2060 (1994)).

In the first series of experiments, examples were presented whereresonance energy transfer (RET) was used to measure cell fusion mediatedby laboratory-adapted strains of the virus (e.g. HIV-1_(LAI)). In thiscase, HeLa cells stably expressing the HIV-1_(LAI) envelope glycoproteinwere mixed with HeLa cells stably expressing human CD4 and the level ofcell fusion measured by RET determinations at intervals followingmixing.

In the first series of experiments, applicants referred to the stableHeLa cell line expressing the envelope glycoprotein of HIV-1_(LAI) asHeLa-env⁺. Applicants will now refer to these cells as HeLa-env_(LAI) ⁺to distinguish them from the HeLa-env_(JR-FL) ⁺ cells described below.

Since the properties of macrophage-tropic strains of HIV-1 differ fromthose of laboratory-adapted strains, applicants have now developed a RETassay to measure membrane fusion mediated by the envelope glycoproteinof a macrophage-tropic HIV-1 isolate. Applicants believe this assaywould have particular advantages for identifying agents which inhibitfusion mediated by macrophage tropic strains of the virus. Such agentsmight differ from those identified using the HeLa-env_(LAI) envelopeglycoprotein RET assay. In the drug screening context, agents whichinhibit fusion mediated by a macrophage-tropic isolate envelopeglycoprotein might be more valuable as lead compounds in the developmentof drugs for the treatment of HIV-1 infection.

As described in more detail below, applicants have constructed a HeLacell line (HeLa-env_(JR-FL) ⁺) which stably expresses the envelopeglycoprotein from the macrophage tropic strain HIV-1_(JR-FL) (Koyanagi,Y. et al., Science 236:819(1987)). RET measurements indicated thatHeLa-env_(JR-FL) ⁺ cells do not fuse with HeLa-CD4⁺ cells or the CD4⁺ Tlymphoblastoid cell line C8166. Applicants then tested HeLa-env_(JR-FL)⁺ cells for ability to fuse with three cell lines that are reportedlysusceptible to infection by macrophage tropic HIV-1 isolates. Theseinclude the T-cell/B-cell hybrid cell line CEM×174 (Stefano, K. A., etal., J. Virol. 67:6707 (1993)), the monocytic leukemia cell line THP-1(Meylan, P. R. A. et al., Virology 193:256 (1993)), and the cell linePM1 which was recently derived from the Hut78 T lymphoblastoid cell line(Lusso, P., et al., J. Virol. 69:3712 (1995)). RET measurements showthat HeLa-env_(JR-FL) ⁺ cells fuse with PM1 cells, but not with CEM×174or THP-1. Fusion of HeLa-env_(JR-FL) ⁺ cells with PM1 cells wasinhibited by agents such as the antibody OTK4A, which inhibits HIV-1attachment. This indicates that fusion measured in this RET assay isspecifically induced by the interaction of the HIV-1_(JR-FL) envelopeglycoprotein with CD4. We have modified the RET assay to make itsuitable for detecting agents which inhibit fusion mediated byHIV-1_(JR-FL). This assay uses the semi-automated microplate formatdescribed as one of two format options in the original application.

Construction of an Expression Vector Encoding HIV-1_(JR-FL) gp120/gp41

The HIV-1_(JR-FL) envelope glycoprotein (gp120/gp41) was expressed usingthe vector pMA243_(JR-FL). This is identical to the vector pMA243 usedto express HIV-1_(LAI) gp120/gp41, except that the HIV-1_(LAI)gp120/gp41 coding sequences were excised from pMA243 and replaced by theHIV-1_(JR-FL) coding sequences. pMA243 is described in Dragic, T., etal., J. Virol. 66:4794 (1992) as well as previously on page 41. pMA243has been deposited with the ATCC as described previously on page 19.

The Detailed Method for Constructing pMA243_(JR-FL) was as Follows

The HIV-1_(JR-FL) envelope sequence was amplified by PCR from theplasmid vector pUCFL112-1 (kindly provided by Dr. I. S. Y. Chen,U.C.L.A., CA) and subcloned into the vector pMA243. Splicing by overlapExtension (SOEing) was used to create the HIV-1_(JR-FL) gp160-dhfr* genesegment. First, the HIV-1_(JR-FL) gp160 sequence was amplified frompUCFL112-1 using primers 1 and 2. Primer 1(5′-ATT-CAG-AAG-AGT-CGC-CAG-AGT-AGA-AAA-GTT-GTG-GGT-CAC-3′) annealed tothe 5′ end of gp160 gene (5′ to the KpnI site) while primer 2(5′-GAT-GGC-ACC-AAG-CTT-ATC-GAT-CTT-ATA-GCA-AAG-CCC-TTT-CCA-AGC-3′)included the antisense strand of the env-dhfr* intergenic region fusedto the complement of the 3′ end of the HIV-1_(JR-FL) gene. Next, thedhfr* sequence was amplified from pMA243 using primers 3 and 4. Primer 3(5′-GAT-CGA-TAA-GCT-TGG-TGC-CAT-CAT-GGT-TCG-ACC-ATT-GAA-CTG--3′)included the sense strand of the env-dhfr* intergenic region fused tothe 5′ end of the dhfr* gene while primer 4(5′-ATG-AGC-CTT-GTG-TGT-GGT-AG-3′) annealed within the 3′-LTR region.The two PCR products were pooled, excess primer removed and a secondround of PCR was performed in the presence of primers 1 and 4. The finalPCR product consisted of the HIV-1_(JR-FL) envelope gene fused to thedhfr* gene. Lastly, the KpnI fragment of pMA243 (encompassing theHIV-1_(LAI) envelope and dhfr* genes) was excised and replaced with theHIV-1_(JR-FL) gp160-dhfr* gene segment. To verify that no mutations wereintroduced by the cloning procedure the KpnI fragment was sequencedusing the dideoxy method. The resultant plasmid has been designatedpMA243_(JR-FL).

Transtection of pMA243_(JR-FL) into HeLa Cells

The cell line HeLa-env-_(JR-FL) ⁺ was made by introducing the plasmidpMA243_(JR-FL) into HeLa cells using the lipofectin procedure (GibcoBRL, Grand Island, N.Y.), following the manufacturer's protocol.Transfectants were selected in 2 μM methotrexate and cloned twice bylimiting dilution. Expression levels of gp120 in HeLa-env_(JR-FL) ⁺ weredetermined by flow cytometry and by an enzyme linked immunosorbent assayfor detecting gp120 shed into the culture supernatant.

RET Assay

Staining of cells was similar to that described in the first series ofexperiments with minor modifications. The modified procedure workseffectively with all cell combinations as described below. Octadecylrhodamine B, chlorine salt (R18) and fluorescein octadecyl ester (F18)(Molecular Probes, Eugene, Oreg.) are dissolved in 100% EtOH to finalconcentrations of 10 mg/ml and 5 mg/ml, respectively. R18 is thendiluted 1:2000 in the appropriate complete tissue culture medium(without selection) and the dye concentration adjusted such that the ODat 565 nm is 0.52±5%. F18 is diluted 1:1000 in complete culture mediumand adjusted such that the OD at 506 nm is 0.34±5%. Medium containingF18 is placed on monolayers of the appropriate envelope-expressing cellline (HeLa-env_(LAI) ⁺ or HeLa-env_(JR-FL)). Medium containing R18 iseither placed on monolayers of the appropriate CD4-bearing cell in thecase of adherent cells (e.g. HeLa-CD4⁺) or added to an equal volume ofmedium containing suspension cells (e.g. PM1 cells). Adherent cells arestained when they are approximately 60-80% confluent. Suspension cellsare stained at a concentration of 0.25-0.5×10⁶/ml. Cells are thenincubated overnight in the fluorescent dye-containing culture media.

Following staining, adherent cells are removed from culture flasks bytreatment with 0.5 mM EDTA and washed several times in culture mediacontaining 10% FBS. Suspension cells are washed by several cycles ofcentrifugation. 20,000 envelope-expressing cells are plated with anequal number of CD4 expressing cells per well of a 96 well plate andincubated for 4 hours at 37 C. Where both cell lines are adherent,flat-bottomed well plates are used. Round-bottomed well plates are usedwhere the CD4-expressing cell is a suspension cell line (e.g. PM1cells). Controls include wells containing each cell line alone.Following three washes in PBS (with low speed centrifugation betweenwashes for the suspension cells), the fluorescence is read in aMillipore Cytofluor plate reader using the filter combinationspreviously described on page 48.

% RET Calculation

The emission values, X, Y and Z are recorded for each cell combination:

A) HeLa or HIV-1 envelope glycoprotein-expressing HeLacells+CD4-expressing cells.

B) HeLa or HIV-1 envelope glycoprotein-expressing HeLa cells alone.

C) CD4-expressing cells alone.

Percent RET is then calculated using the formula previously described onpage 49.

Experimental Results

% RET results obtained using this assay with various cell combinationare given in Table 2. As shown in Table 2, HeLa-env_(LAI) ⁺ cells fusereadily with HeLa-CD4⁺ cells (% RET value >5). Similarly, HeLa-env_(LAI)cells fuse readily with the CD4⁺ T lymphoblastoid cell line C8166. Inboth cases fusion is inhibited to background levels (% RET values <1) bythe antibody OTK4A (Ortho Diagnostic Systems, Raritan, N.J.). OKT4A isan anti-CD4 antibody which is known to block the binding of gp120 toCD4, the attachment step of viral entry and an essential prelude toHIV-1 envelope-mediated cell fusion. HeLa-env_(LAI) ⁺ cells do not fusewith chinese hamster ovary cells expressing CD4 (CHO-CD4⁺), sincenon-primate cells appear to lack accessory molecules that are requiredfor HIV-1 envelope-mediated cell fusion.

TABLE 2 RET measurements obtained using various combinations of cellsand the inhibitory antibody OKT4A (0.3 μg/ml). F18 R18 labeled cellslabeled cells Inhibitor % RET HeLa-env_(LAI) ⁺ HeLa-CD4⁺ none 7.3HeLa-env_(LAI) ⁺ HeLa-CD4⁺ OKT4A 0.4 HeLa HeLa-CD4⁺ none 0.7HeLa-env_(LAI) ⁺ C8166 none 13 HeLa-env_(LAI) ⁺ C8166 OKT4A 0.6 HeLaC8166 none 0.5 HeLa-env_(LAI) ⁺ CHO-CD4⁺ none 0 HeLa-env_(JR-FL) ⁺HeLa-CD4⁺ none 0.8 HeLa-env_(JR-FL) ⁺ C8166 none 0 HeLa-env_(JR-FL) ⁺CEMx174 none 0 HeLa-env_(JR-FL) ⁺ THP-1 none 0 HeLa-env_(JR-FL) ⁺ PM1none 7.5 HeLa-env_(JR-FL) ⁺ PM1 OKT4A 0.7 HeLa PM1 none 0.3

As shown in Table 2, HeLa-env_(JR-FL) ⁺ cells do not fuse with HeLa-CD4⁺or C8166 cells as indicated by the background levels of % RET (% RET<1). In this regard, the HeLa-env_(JR-FL) ⁺ cells mimic HIV-1_(JR-FL)which is macrophage tropic. Like other macrophage-tropic HIV-1 isolates,HIV-1_(JR-FL) generally does not fuse with or infect T lymphocyte celllines or other CD4⁺ cell lines (O'Brien, W. A. et al., Nature 348:69(1990)).

Next, applicants tested several cell lines which have been reported tobe infectable by macrophage-tropic strains of HIV-1 as described above.Applicants found that HeLa-env_(JR-FL) ⁺ cells did not fuse with CEM×174or THP-1 cells in the RET assay (Table 2). Applicants also tested fusionbetween HeLa-env_(JR-FL) ⁺ cells and the PM1 cell line. These cells wereobtained from M. Norcross (FDA, Bethesda, Md.) and M. Crowley (NIH,Bethesda, Md.). As shown in Table 2, HeLa-env_(JR-FL) ⁺ cells and PM1cells did fuse as determined by % RET measurements. The time course offusion between HeLa-env_(JR-FL) ⁺ cells and PM1 cells, measured usingthe RET assay, was similar to that seen with the HeLa-env_(LAI) ⁺ andHeLa-CD4⁺ cell combination (FIG. 5). Note that the results usingHeLa-env_(LAI) ⁺ and HeLa-CD4⁺ cells are similar to, but not identicalwith, those presented in FIG. 1. The minor differences may result fromthe use of the plate reader assay method in FIG. 5 rather than thecuvette assay method in FIG. 1.

The specificity of fusion between HeLa-env_(JR-FL) ⁺ and PM1 cellsmeasured using the RET assay is supported by the lack of fusion(background % RET measurements) obtained when HeLa-env_(JR-FL) cellswere mixed with other CD4⁺ cells (Table 2 and above). Moreover, onlybackground levels of % RET were found when PM-1 cells and HeLa cellswere mixed in the assay (Table 2). Finally, fusion between PM-1 cellsand HeLa-env_(JR-FL) ⁺ cells, measured by the RET assay, was assensitive to inhibition by OKT4A as was fusion between HeLa-env_(LAI) ⁺cells and HeLa-CD4⁺ cells (FIG. 6). RET was completely inhibited tobackground levels by 0.3 μg/ml OKT4A (Table 2). No inhibition of RET wasseen using the same concentration of the antibody OTK4 (Ortho DiagnosticSystems), which does not inhibit HIV-1 attachment (not shown).

Third Series of Experiments

Inibition of HIV-1 envelope glycoprotein-mediated membrane fusion in theRET assay by anti-PM1 hybridoma supernatants

The RET assay described in this application is useful for screeningantibodies capable of inhibiting HIV-1 envelope glycoprotein-mediatedmembrane fusion. Hybridomas against PM1 cells were generated and thesupernatants from these hybridomas were screened in the RET assay toidentify hybridomas which secret antibodies capable of inhibiting fusionbetween HeLa-env_(JR-FL) and PM1 cells. The culture supernatants fromhybridomas PA-3, PA-5, PA-6 and PA-7 inhibited fusion betweenHeLa-env_(JR-FL) and PM1 cells in the RET assay, and also inhibitedfusion between HeLa-env_(LAI) cells and certain CD4⁺ target cells (Table3). HIV-1_(LAI) envelope glycoprotein-mediated membrane fusion with PM-1and HUT-78 was inhibited by all of the mAb secreted from these hybridomacell lines. Whereas, fusion between HeLa-env_(LAI) and CEM was inhibitedby PA-3 and PA-5 but less so by PA-6 or PA-7. The fusion betweenHeLa-env_(LAI) and C8166 or Sup-T1 cells was inhibited minimally or notat all by these mAb.

TABLE 3 Inhibition of HIV-1 envelope glycoprotein mediated cell fusionby novel mAb. Envelope % Inhibition of RET by expressing CD4⁺ novel mAbcells cell % RET PA-3 PA-5 PA-6 PA-7 HeLa-env_(JR-FL) PM-1 16.3 85.396.3 92 67 HeLa-env_(LAI) PM-1 12.4 89.7 100 81 69 HeLa-env_(LAI) HUT-7810.9 51.3 60.3 55.7 52.7 HeLa-env_(LAI) CEM  9.5 71.8 68 33 21HeLa-env_(LAI) HeLa-CD4 11.4  0 0  7.7  0 HeLa-env_(LAI) SUP-T1 19.8 2.5 0 18 11 HeLa-env_(LAI) C8166 15.4  9.7 22 22.3 13

Effect of β-chemokines on HIV-1 envelope glycoprotein-mediated membranefusion analysed by the RET assay

The RET assay was developed further to analyze fusion between cellsexpressing the HIV-1 envelope glycoprotein and primary CD4⁺ cells. CD4⁺target cells (mitogen-activated primary human CD4⁺ lymphocytes, primaryhuman macrophages or PM1 cells) were labeled with octadecyl rhodamine(Molecular Probes, Eugene, Oreg.), and Hela-env_(JR-FL) cells,HeLa-env_(LAI) cells (or control HeLa cells, not shown) were labeledwith octadecyl fluorescein (Molecular Probes), overnight at 37° C. Equalnumbers of labeled target cells and env-expressing cells were mixed in96-well plates and β-chemokines (or the CD4 MAb OKT4a) were added at thefinal concentrations (ng/ml) indicated in parentheses in the firstcolumn of Table 4.

Fluorescence emission values were determined 4 h after cell mixing. RETand percentage inhibition of RET were calculated as described above. Inthis experiment, HeLa-env_(JR-FL) fused with CD4⁺ normal humanT-lymphocytes (% RET=6.0) and macrophages (% RET=4.3) as well as PM1cells (% RET=11.5). HeLa-env_(LAI) also fused with CD4⁺ normal humanT-lymphocytes (% RET =10.5) and PM1 cells (% RET=10.5) but not withmacrophages (% RET=1.2, similar to % RET using HeLa cells in place ofHeLa-env cells).

TABLE 4 Inhibition of membrane fusion by β-chemokines % Inhibition ofRET HeLa-env_(JR-FL) HeLa-env_(LAI) a) PM1 cells no chemokines 0 0+R/Mα/Mβ (80/400/100) 99 5 +RANTES (80) 92 0 +MIP-1α (400) 61 0 +MIP-1β(100) 87 7 +MCP-1 (100) 1 2 +MCP-2 (100) 28 7 +MCP-3 (100) 2 1 b) LW5CD4⁺ cells no chemokines 0 0 +R/Mα/Mβ (106/533/133) 61 0 +RANTES (106)35 5 +MIP-1α (533) 28 0 +MIP-1β (133) 56 8 +OKT4A (3 ug/ml) 100 100 c)EU2 CD4⁺ cells no chemokines * 0 +R/Mα/Mβ (320/1600/400) * 0 +RANTES(320) * 0 +MIP-1α (1600) * 0 +MIP-1β (400) * 0 d) Macrophages nochemokines 0 * +R/Mα/Mβ (80/400/100) 54 * +RANTES (80) 20 * +MIP-1α(400) 15 * +MIP-1β(100) 37 * +MCP-1 (100) 0 * +MCP-2 (100) 28 * +MCP-3(100) 18 * *No detectable fusion

As shown in Table 4, RANTES, MIP-1β (and to a lesser extent, MlP-1α)strongly inhibited membrane fusion of HeLa-env_(JR-FL) cells with PM1cells, whereas fusion between PM1 cells and HeLa-env_(LAI) cells wasinsensitive to these β-chemokines (Table 4a). Similar results wereobtained with primary CD4⁺ T-cells from a normal laboratory worker (LW5,Table 4b), although higher concentrations of β-chemokines were requiredto inhibit membrane fusion in the primary cells than in PM1 cells. Thus,the actions of the β-chemokines are not restricted to the PM1 cell line.In marked contrast to LW5's cells, CD4⁺ T-cells from an exposed butuninfected individual (EU2) did not fuse with HeLa-env_(JR-FL) cells (%RET=0.1), whereas they could clearly fuse with HeLa-env_(LAI) cells (%RET=4.1) in a β-chemokine-resistant manner (Table 4c). Fusion betweenHeLa-env_(JR-FL) and primary macrophages was inhibited only weakly bythe β-chemokines, while HeLa-env_(LAI) did not fuse with primarymacrophages (Table 4d). The RET assay demonstrates that β-chemokinesinterfere with env-mediated membrane fusion. It also establishes thatenvelope glycoproteins from a primary, NSI strain cannot fuse with CD4⁺T-cells from an EU individual, providing a critical clue to how thesecells may resist HIV-1 infection in vitro, and perhaps in vivo.

What is claimed is:
 1. A method for determining whether an agent iscapable of specifically inhibiting (A) the fusion of a macrophage-tropicprimary isolate of HIV-1 to a CD4⁺ cell susceptible to infection by amacrophage-tropic HIV-1 or (B) the fusion of a T cell-tropic isolate ofHIV-1 to a cell susceptible to infection by a T cell tropic HIV-1, butnot both, which comprises: (a) contacting (i) a first appropriate CD4⁺cell, which is labeled with a first dye, with (ii) a cell expressing theHIV-1 envelope glycoprotein of the macrophage-tropic primary isolate ofHIV-1 on its surface, which is labeled with a second dye, in thepresence of an excess of the agent under conditions which would normallypermit the fusion of the CD4⁺ cell to the cell expressing the HIV-1envelope glycoprotein on its surface in the absence of the agent, thefirst and second dyes being selected so as to allow resonance energytransfer between the dyes; (b) exposing the result of step (a) toconditions which would result in resonance energy transfer if fusion hasoccurred; and (c) determining whether there is a reduction of resonanceenergy transfer, when compared with the resonance energy transfer in theabsence of the agent; (d) contacting (i) a second appropriate CD4⁺ cell,which is labeled with a first dye, with (ii) a cell expressing the HIV-1envelope glycoprotein of a T cell-tropic isolate of HIV-1 on itssurface, which is labeled with a second dye, in the presence of anexcess of the agent under conditions which would normally permit thefusion of the CD4⁺ cell to the cell expressing the HIV-1 envelopeglycoprotein on its surface in the absence of the agent, the first andsecond dyes being selected so as to allow resonance energy transferbetween the dyes; (e) exposing the result of step (d) to conditionswhich would result in resonance energy transfer if fusion has occurred;and (f) determining whether there is a reduction of resonance energytransfer, when compared with the resonance energy transfer in theabsence of the agent, wherein a decrease in transfer in step (c) but notstep (f) indicates that the agent is capable of specifically inhibitingfusion of the macrophage-tropic primary isolate of HIV-1 to CD4⁺ cellsand a decrease in transfer in step (f) but not step (c) indicates thatthe agent is capable of specifically inhibiting the fusion of a Tcell-tropic isolate of HIV-1 to the CD4⁺ cells.
 2. The method of claim1, wherein the first appropriate CD4⁺ cell is a PM1 cell, a primaryhuman T lymphocyte, or a primary human macrophage.
 3. The method ofclaim 1, wherein the second appropriate CD4⁺ cell is a HeLa-CD4 cell, aprimary human T lymphocyte, a human T cell line, PM1 cell, or a C8166cell.
 4. The method of claim 1, wherein in step (a) the cell expressingthe HIV-1 envelope glycoprotein of the macrophage-tropic primary isolateis an HIV-1_(JR-FL) gp120/gp41 HeLa cell.
 5. The method of claim 1,wherein in step (d) the cell expressing the HIV-1 envelope glycoproteinof the T-cell-tropic of HIV-1 is an HIV-1_(LAI) gp120/gp41 HeLa cell. 6.The method of claim 1 wherein the agent is not previously known.
 7. Themethod of claim 1, wherein the first dye is a rhodaminemoiety-containing molecule and the second dye is a fluoresceinmoiety-containing molecule.
 8. The method of claim 7, wherein therhodamine moiety-containing molecule is octadecyl rhodamine B chlorideand the fluorescein moiety-containing molecule is fluorescein octadecylester.
 9. The method of claim 1, wherein the first dye is a fluoresceinmoiety-containing molecule and the second dye is a rhodaminemoiety-containing molecule.